High-throughput cell transfection device and methods of using thereof

ABSTRACT

Transfecting biology cells with nucleic acid molecules (DNA, siRNA) is an essential prerequisite in elucidating how genes function in complex cellular context and how their activities could be modulated for therapeutic intervention. Traditionally studies are carried out on a low throughput gene-by-gene scale, which has created a huge bottleneck in functional genomic study and drug discovery. Development of high-throughput cell transfection technology will permit functional analysis of massive number of genes and how their activities could be modulated by chemical or biological entities inside cells. This invention describes design, construction of device and apparatus for high throughput effective cell transfection. Procedures and protocols for using the device and apparatus are also described in the application. Novel methods of using the device in cell-based assays are also disclosed.

CROSS-REFERENCE

Not Applicable.

FIELD OF THE INVENTION

This invention relates to the biotechnology field in general and withspecific reference to effective transfection of biological cells in ahigh throughput fashion, and its applications in drug discovery anddevelopment.

BACKGROUND OF THE INVENTION

Being able to effectively introduce foreign molecules, such as DNA,siRNA, protein, etc., into biological cells is of great importance inbiology and biotechnology. Particularly effective transfection ofbiological cells with nucleic acid molecules is an essentialprerequisite in elucidating how genes function in complex cellularcontext and how their activities could be modulated for therapeuticintervention. Cell transfection is routinely used in fundamental biologyresearch and pharmaceutical development. Traditionally studies arenormally carried out on a low throughput gene-by-gene scale, which hascreated a huge bottleneck in functional genomic study and drugdiscovery. Development of high-throughput cell transfection technologywill permit functional analysis of massive number of genes and how theiractivities could be modulated by chemical or biological entities.Development of high-throughput cell transfection methods willsignificantly accelerate biology research and facilitate translation ofgenomic knowledge into therapeutic means in fighting various diseases.

Several methods are currently available for cell transfection, such asviral transduction, lipofection, electroporation, etc. Recombinantvectors derived from viruses are very effective in transfectingengineered cell lines and primary cells. However, use of viral vectorsoften results in undesirable alteration of cellular functions, inaddition vector preparation is time-consuming and laborious, thus itsapplication in high-throughput transfection is limited. Lipid basedtransfection methods are routinely used in high-throughput transfectionapplications, and a recent invention based on lipid transfection claimsultra-high-throughput capability [U.S. Pat. No. 6,544,790].Nevertheless, all lipofection methods lack of ability of transfectionnon-dividing cells, particularly primary cells directly derived fromanimal tissue.

Electroporation is a process associated with transient permeabilizationof cell membranes under electrical fields. It has been shown to becapable of delivering various substances (genes, siRNAs, antibodies,proteins and nanoparticles) into virtually any type of cells (engineeredcell lines and primary cells). On the other hand, electroporation isoften known for low efficiency, poor inconsistency, and extensive celldamage. This is largely due to the trial-and-error approach adopted byconventional electroporation systems, which apply hundreds to thousandsof volts to cells suspended in solution, inevitably kill large portionof cells due to a process called irreversible electroporation.

Several novel methods and devices have been invented recently to addressissues of low transfection efficiency and poor cell motility associatedwith electroporation [U.S. Pat. No. 6,300,108, U.S. Pat. No. 6,403,348,US2005/0170510]]. The related arts employ feed-back mechanisms tomonitor electroporation in cells so to achieve high degree oftransfection efficiency and high cell motility. The new methods anddevices have been proven to be very useful in transfection of cells witha variety of foreign molecules. However, the design of the devices limittheir capability of processing cells in high-throughput fashion, whichis particularly required by cell-based assays for functional genomicsstudy and drug discovery. Secondly, in the case of transfecting cellswith DNA molecules, the related arts do not address effective deliveryof DNA molecules into cell nuclei, which is required for cells toexpress proteins encoded by the DNA molecules. These and other needs areaddressed by the transfection devices of the present invention. Inaddition, novel use of the disclosed transfection devices and methods incell-based assays employing transient transfection is also presented inthis invention.

BRIEF DESCRIPTION OF THE INVENTION

The present invention includes a high-throughput cell transfectiondevice, methods for making and using the cell transfection device. Thecell transfection device has two chambers separated by a thin porousmembrane where cells can attach. Proper differential electric potentialis generated across the cells residing on the porous membrane toelectroporate the cells, thus permitting effective delivery of foreignmolecules into the cells. The porous membrane can be treated to allowselective transfection of cells at particular location, leaving the restof cells unaffected. A novel approach is also presented that permitsrealization of high-density electrical connections through convex liquiddroplets, as well as means to form liquid droplets at selectedlocations. Methods for introducing foreign molecules to the cells in ahigh-throughput fashion are also presented. With the ability oftransfecting cells in a high-throughput mode, we further describemethods that employ transfection of cells for profiling transcriptionfactor activities in cells, and profiling influence of transporterproteins on transportation of molecules in and out of cells, and furthertheir applications in drug discovery.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures:

FIG. 1 is a schematic cross-section view of a traditional cell cultureinsert for growing cells on a porous membrane.

FIG. 2 is a schematic cross-section view of a novel cell culture devicefor growing cells on a porous membrane.

FIG. 3 shows a typical method of constructing the cell culture device ofFIG. 2.

FIG. 4 is two schematic illustrations of a high-throughput celltransfection device containing an array of cell transfection units, andcross-section view of a typical cell transfection unit

FIG. 5 is a schematic view of a plate containing an array of electrodepads.

FIG. 6 is three schematic illustrations of establishing electricalconnections via electrical conductive liquid droplets.

FIG. 7 is three schematic illustrations of a method for parallel loadingof foreign molecules to a cell transfection device by pre-depositing themolecules on electrode pads.

FIG. 8 is two schematic illustrations of parallel reagent dispensingthrough probe electrodes.

FIG. 9 is a schematic illustration of a means to connect electrodes byrow and column.

FIG. 10 is a schematic illustration of the method for measuring cellconfluency

FIG. 11 is three schematic illustrations of effect of ununiformity incell attachment on electrical currents flowing through a porous membranewith cells, and a design to overcome the effect.

FIG. 12 is a schematic diagram of a typical cell transfection apparatus

FIG. 13 is a schematic demonstration of two methods for improvingdelivery of DNA molecules into cell nuclei.

FIG. 14 is a schematic illustration of using auxiliary medium feedingplates to add more culture media to cells grown in the cell culturedevice in FIG. 2

FIG. 15 is two schematic illustrations of a method for parallel cellplating

FIG. 16 is two schematic illustrations of using flexible auxiliarymedium feeding plate to add more culture media

FIG. 17 is an image of MDCK cells stained by YOYO-1 fluorescent dye

FIG. 18 is two images showing effective DNA transfection in two primarycell lines

FIG. 19 is a image of PrEC cells transfected with labeled siRNA

FIG. 20 is two images showing effective knockdown of GFP in PrEC cellsby effective siRNA transfection

FIG. 21 is two images showing selective electroporation and transfectionin MDCK and PrEC cells

DETAILED DESCRIPTION OF THE INVENTION

Before the present methods, treatments and devices are described, it isto be understood that this invention is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acompound” includes a plurality of such compounds and reference to “thesample” includes reference to one or more samples and equivalentsthereof known to those skilled in the art, and so forth.

Cell Transfection Device and Apparatus Cell Culture Device Design andConstruction

The cell transfection device described in the present invention isintended to transfect cells attached to a porous membrane, in ahigh-throughput fashion. To culture cells on a porous membrane, itrequires feeding cells from both side of the porous membrane.Traditional ways to culture cells on a porous membrane involves use ofcell culture insert (1) and a separate companion well (2), as depictedin FIG. 1. Cells (3) are introduced to the insert that has a porousmembrane (4) where cells can attach. The drawback of this design is thatit is difficult and costly to construct high-throughput device whichnormally contains more than 96 individual units on the industrialstandard 5″×3.3″ microtiter plate format. In the present invention, acell culture device is depicted in FIG. 2. Unlike the conventional cellculture inserts, the described cell culture device has a first chamber(1), and a second chamber (2), and a porous membrane (3) separating thetwo chambers. Cells (4) can be introduced to any one of, or bothchambers, and attach to either side, or both sides, of the porousmembrane. It should be noted the simple geometry of the openings shownin FIG. 2 is for illustration, actual device can have more complexopenings to serve specific purposes.

Because of the simplicity of the device, it is straight forward tomultiply such devices in an array format for high-throughputapplications. FIG. 3 illustrates a type construction process, a 24×16array of openings in 384-well microplate footprint is presented forillustration purpose. A porous membrane (1) is first attached, usingthermal welding or gluing, to a first rigid support plate (2) with anarray of openings. The porous membrane is made of dielectric materials,including but not limited to, polycarbonate, PET, etc., with a filmthickness from 1um to 1 mm. The support plate is made of rigid anddielectric materials including but not limited to, plastic, glass,quartz etc., and is from 0.5 mm to 10 mm in thickness. Thecharacteristic dimension of the openings of the support plate is between0.1 mm to 10mm. Then a second plate (3) with the same number of openingsthat are individually aligned with the openings of the first plate, isattached to the other side of the porous membrane, thus producing a cellculture plate with an array of cell culture devices depicted in FIG. 2.The second plate is also made of dielectric materials. It should benoted that the second plate can be substituted with a thin dielectricfilm with openings, or dielectric adhesive paste applied to the specificarea of the porous membrane to expose specific area of the porousmembrane under the openings of the first support plate.

Cell Transfection Device Design

The described cell transfection device employs electroporation topermeabilize cells so that nucleic acid molecules (DNA, siRNA) or othermembrane-impermeable macromolecules present in the cell media can enterthe cells. FIG. 4.a illustrates a typical cell transfection device,which consists of three components, one cell culture plate (1) aspreviously described, two electrode-containing plates (2, 3) placed oneach side of the cell culture plate, which are used to generate adequateelectrical potentials across the cells residing on porous membrane toinduce electroporation. The electrode-containing plates have electricalconnections (4) through which the electrodes can be accessed by outsideelectronics. Typically a transfection device contains an array ofindividual units (FIG. 4 b) (a 24×16 array of transfection units areshown in FIG. 4 a), cells residing in each units can be transfectedseparately.

In a typical process, cells (5) are first grown on the porous membrane(6) inside each cell culture unit (7), as shown in FIG. 4 b, followed byintroducing foreign substances (8), such as DNA, siRNA, protein, etc.,to one of the two chambers (9,10) that are filled with electricalconductive media. Proper differential electrical potential is createdbetween the two electrodes (11,12) to electroporate the cells withoutinducing cell damage due to irreversible membrane breakdown. Foreignsubstances then can enter the electroporated cells either by passivediffusion, or electrophoresis in the case of molecules with net charges,or both.

Electroporation Electrode Design

The electrode plate contains an array of electrodes used to applyelectrical field to individual cell culture units. FIG. 4 b shows use ofprobe electrodes (11,12) that have smaller size than the openings of thechambers (9,10) of the cell culture unit. The probe electrodes can bepositioned close to cells so that low voltage (0.5V to 10V) can beapplied to electroporate the cells. The drawback of using probeelectrodes is that since the size of the electrodes are smaller than thesize of the porous membrane, only the cells directly underneath theelectrodes are electroporated. In addition, construction of ahigh-density probe electrode array can be expensive.

FIG. 5 illustrates a electrode plate (1) containing an array of padelectrodes (2) that can be made at high density using techniques such asprint circuit board techniques, at relatively low cost. The surfaces ofthe electrodes are rendered to be hydrophilic, and their surroundingareas (3) are rendered to be hydrophobic, for the purpose to bediscussed below. The electrode pads are made of electrical conductivematerials, including but not limited to, thin film metal, conductivecomposite materials, conductive gel or paste, or a combination to meetspecific needs.

Establish Electrical Contact via Liquid Droplets

In constructing high-throughput electroporation mediated celltransfection device, one of the major challenges is to implementhigh-density transfection units at reasonable cost without electricalcross talking and biochemical contamination among individual units. Amethod is hereby described to overcome this challenge by using a unique“aqueous contact” approach.

FIG. 6 illustrates the method for establishing electrical connectionsthrough convex droplets of electrical conductive media. Similarly as tothe electrode plate, the cell culture plate is also treated to rendersurfaces (1) of inner walls of the cell culture chambers hydrophilic,and the rest of the surfaces (2) hydrophobic. With such treatment, whenadequate amount of conductive liquid is added into the chambers of eachcell culture unit (FIG. 6 a), convex aqueous droplets (3) can be formeddue to surface tension force. Furthermore, the droplets are confined tothe openings of each individual unit because of the hydrophobic coatingaround the openings that prevent the droplets from spreading, thuseliminating cross-contamination among individual units. Using the sameapproach, aqueous droplets (3) are also formed atop of each electrodepad that has a hydrophilic surface (4), similarly, hydrophobic coating(5) around electrode pads is used to confine movement of droplets aspreviously discussed. After forming aqueous droplets (FIG. 6 b), theelectrode plates and the cell culture plates are brought close to letthe droplets in contact and form stable aqueous plugs (6), which againare retained and contained due to surface tension force (FIG. 6 c).Because both cell growth and electroporation media are electricallyconductive, the confined aqueous plugs form electrical connectionsbetween each pair of corresponding electrodes without electrical crosstalking and biochemical contamination. Using this approach, it isfeasible to build high-density electroporation array, for example tocomfort to 384-well or 1536-well industry standard high-throughputfootprints.

Introduction of Nucleic Acid Molecules

In electroporation mediated cell transfection, nucleic acid moleculesare usually mixed in electroporation solution and then added to cellmedia by manually pipetting or through use of automated liquiddispensing apparatus. This traditional approach adds additional time forsample preparation and liquid handling, which could be significant inhigh-throughput scenario.

To circumvent this step, nucleic acid molecules can be introduced tocell transfection device before cells are plated. In the case that padelectrodes are used, DNA or RNA molecules can be pre-deposited to eachindividual electrode (1) on one of the electrode-array plates (2) (FIG.7 a), then let the solution dry to leave the nucleic acid molecules (3)on the electrode (FIG. 7 b). This step can be realized usingcommercially available microarray spotters that are capable ofdepositing different or the same DNA and RNA molecules in high-densityand high-throughput. The nucleic acid molecule coated plate can bestored for extended period of time for later use. When cells are readyfor transfection, electroporation media will be added to each electrodesas previously described, then the nucleic acid molecules attached to theelectrode surface are released into the droplet (4) and brought to incontact with cells when aqueous contacts are formed (FIG. 7 c). Thisapproach also provides flexibility on where to load transfectionreagents to the cells grown on one side of porous membrane, whether fromthe apical side (the side of cells in full contact with media) or thebasal side (the side of cells in contact with porous membrane surface),by simply flipping the position (top or bottom) of the electrode-arrayplate that has DNA/RNA on it. This can be very useful when transfectingcertain types of polarizable cells, which when form a polarizedmonolayer, exhibit different DNA/RNA uptake behaviors between the basaland apical sides of the monolayer. This method can also be used tointroduce other molecules such as protein, peptide, etc.

In the case that probe electrodes are used, the electrode probes canalso be used to load transfection reagents such as nucleic acidmolecules, as shown in FIG. 8. To achieve this purpose, one can treatthe surface of the electrode probes (1) such that liquid droplets (2)can be formed at the tips of the probe electrodes due to surface tensionforce. Alternatively, one can cut a groove (3) near the tip of a probeelectrode, and the groove can serve as a reservoir for solutionscontaining foreign substances such as nucleic acid molecules (4). Toload foreign molecules to the probe electrodes, the probes (1) arefirstly immersed in wells (5) that contain the same or different DNAsolutions. After retracting the probes, small amount of reagent solutionwill remain on each probe, which can then be introduced to cells whenthe probes are immersed in cell culture devices (6). To speed up mixingof reagent in the cell culture/transfection media inside cell culturedevices, small displacement vibration can be applied to the probes orthe plate (7) that hold the probes. This vibration can also be appliedduring electroporation to reduce the possibility of aggregation ofelectrochemical products (such as gases) on the surface of theelectrodes, which could deteriorate the surface electrochemicalproperties of the electrodes.

Electrode Connection and Application of Electrical Pulses

In the case that a cell transfection device contains an array ofindividual cell transfection units, it is required to be able toelectrically activate every pair of electrodes that causeselectroporation of the cells in the corresponding unit, in order tooptimize electroporation for cells in that unit. Our device is designedto meet this requirement with minimal requirements of wiring, whichcould be difficult and costly to implement in high-throughput devices.To achieve this, the electrodes on the two electrode-array plates areconnected by row and column respectively. FIG. 9 illustrates electricalconnections for a 96-unit in 8(row)×12(column) format. Duringelectroporation process, only one row (i) of electrodes on one electrodeplate is activated, similarly, only one column (j) of electrodes on theother electrode plate is simultaneously activated, which result in onlyactivating the transfection unit that is in both row i and column j(Unit_(ij)). By repeatedly activating the rest row and column ofelectrodes on the two plates, the entire array of units can beindividually activated. Using this configuration, only 20 connections (8rows and 12 columns) are required to be able to address every unit. For384-well plate format that has 16 rows and 24 columns of units, only 40connections are needed in order to activate electroporation on eachindividual unit, comparing to 798 (2 times of 384) wires if eachelectrodes are individually wired and connected to outside electronics.

Electroporation Optimization

Successful electroporation depends on many parameters. In the case ofusing the device disclosed in the present invention, the two mostinfluential parameters are cell type and confluent state of cellmonolayer. When cells are the same, the higher confluency of the cells(means more areas of the substrate cells grow on are covered by cells),the less intense electrical pulses are needed to electroporate thecells. The confluent state of the cell monolayer can be measuredelectrically by applying a low amplitude electrical pulse between twoelectrodes (FIG. 10). Because the pulse doesn't induce electroporationin cells (1), the electrical current through normal cells is negligible.Thus, the electrical current flowing between the two electrodescomprises two component, i.e., leakage current (2) that passes throughthe areas of the porous membrane not covered by cells, and paracellularcurrent (3) that flows between the gaps among cells and between cell andporous membrane substrate. The leakage current is directly determined bycell confluency since the higher the cell confluence, the less number ofpores not covered by cells, thus the larger electrical currents can flowwhen the same electrical potential is applied between the electrodes. Inmost cases, the paracellular current is much smaller than the leakagecurrents, thus leakage current can be approximated with the electricalcurrent flowing between the electrodes, and the state of cell confluencycan be estimated. With the information on cell confluency and cell type,electrical pulses can be optimized to achieve high degree ofelectroporation while avoiding excessive cell damage. A computer programcan be used to determine parameters (amplitude, length and shape) ofelectrical pulses based on theoretical calculation, experimental data,or the combination, to achieve optimal electroporation for the giventype of cells in a given confluent state (measured by electricalcurrents under a constant measurement pulse).

Minimize Effect of Cell Growth Ununiformity

When electroporating a cell monolayer grown on top of a porous membraneusing long electrical pulses, it is important that the porous membraneis effective covered by the cell monolayer, which results in maximaldegree of electroporation effectiveness under the same electricalpulses. However, as illustrated in FIG. 11 a, most times cells don'tcover the entire substrate they grow on, particularly toward the edge ofa chamber where there are always large patches of openings (1) notcovered by cells, even cells reach a high degree of confluency in thecentral areas (2). The exposed patches of porous membrane at the edge ofthe chamber often cause less effectiveness in cell electroporation asthey allow a substantial amount of leakage current (3) passes directlythrough the porous membrane (FIG. 11 b), instead of through the cellmonolayer as effective electroporation Current (4) that forces cellmembranes to be permeabilized.

To overcome this edge effect in cell growth, one can block the pores inthe edge region of the porous membrane, thus minimize leakage currentthat otherwise can flows through the uncovered pores. This concept canbe readily realized by making the opening of the top chamber (5), inwhich cells are cultured, larger than that of the bottom chamber (6), asshown in FIG. 11 c. By this means, electrical current can only flowthrough the central region of the porous membrane/cell monolayer, whichis defined by the opening of the bottom chamber, thus effectivelyeliminating leakage current through the edge of porous membrane/cellmonolayer.

Selective Cell Electroporation and Transfection

Another important benefit arising from the device described in FIG. 11 cis the ability of realize electroporation, and thus transfection, in aselective fashion. Using the method and device in the present invention,a cell can be electroporated only when it is exposed to at least oneunobstructed microscale pore, which permits passage of electroporationcurrent through the cell. If there is no pore beneath the cell or thepores are blocked, the cell will not be affected by the presentelectrical field. Therefore, blocking pores in specific region of theporous membrane provide an effective means to achieve selectiveelectroporation and transfection. In the device shown in FIG. 11 c, thesmaller chamber also defines an electroporation window in the centralregion of the porous membrane/cell monolayer, with cells inside thewindow, or cells directly on top of the bottom chamber opening, can beelectroporated. The rest of the cells outside of this central windowwill not be electroporated and thus transfected since their underneathpores are effectively blocked by the dielectric body of the bottomchamber. The unaffected cells can serve as negative control cells, whichare very useful to study side-by-side how the transfected cells differfrom the unaffected ones due to incorporating of foreign molecules. Toachieve selective electroporation with more sophisticated pattern, onecan use techniques such as glue spotting, screening printing, etc., toprecisely blocked pores at desirable areas of the porous membrane.

Cell Transfection Apparatus

A cell transfection apparatus (FIG. 12) typically comprises a celltransfection device as previously described (1), an electroporationcontrol apparatus (2) and a computer (3). The computer can be either apersonal computer linked with the control apparatus or a microprocessorsystem embedded in the apparatus. The computer determineselectroporation conditions for a particular transfection unit on thecell transfection device, the information is then directed to thecontrol apparatus that selects and drives a pair of electrodescorresponding to a particular transfection unit to achieve optimalelectroporation in the cells residing in the device.

Improving Nuclear Importation of DNA Molecules

In transfecting cells with DNA molecules, the DNA molecules (mostly DNAplasmids) need to enter cell nucleus in order to be expressed. This canbe a challenging issue in transfecting non-dividing cells as unlike individing cells foreign genes can be incorporated in the nuclei ofdaughter cells during mitosis, genes in the cytoplasms of non-dividingcells need to find a way to enter nuclei, otherwise they will be quicklydegraded by cytoplasmic nucleases.

There are a couple of techniques to improve the efficiency oftransporting DNAs from cytoplasm to nucleus, which when combined withour electroporation method can produce better gene transfectionefficiency. One technique involves incorporating DNA nuclear targetingsequence (DTS) (1) in DNA plasmid vectors (2), which improves nuclearlocalization of the DTS-containing DNA molecules through piggy-backingwith DTS-binding proteins (3) that can enter cell nucleus (FIG. 13.a).Another method involves attaching nuclear localization signal (NLS)peptide (4) to DNA plasmids, the NLS peptide can directly modulateproteins (5), such as importins, that control the traffic throughnuclear envelope, so that the NLS-attached DNA plasmids can enternucleus through nuclear pore complexes (FIG. 13.b)

Auxiliary Component for Culturing Cells

The transfection method described in this invention requires cells togrow into a relatively confluent monolayer before they can beelectroporated and transfected. This normally takes a couple of days.Such long culture time requires sufficient cell growth media to keepcells healthy, which is an issue in culturing cells on high-throughputplates as their wells are small in volume and liquid evaporation is abig issue at this physical scale. One approach to solve this problem tois change media from time to time manually or using robotic media changestation, a tedious and time-consuming procedure. As an alternativeapproach, we disclose two methods for increasing culture mediaaccessible to cells in each cell culture unit.

FIG. 14 illustrates one approach that uses specially designed detachablecell culture medium feeding plates. The feeding plates (1) have array ofliquid reservoirs that are aligned with every individual transfectionunit of a cell culture plate (2) where cells reside. The thickness ofthe plates can range from 1 mm to 50 mm depending on how much mediumeach reservoir is expected to accommodate. The reservoirs' inside andthe vicinity of their openings are treated to be hydrophilic (3) and therest areas (4) are rendered to be hydrophobic so that when adequateamount of liquid added to each reservoir, convex liquid droplets (5) canform at the openings of each reservoir. Loading reservoirs with cellculture media can be done using pipetting techniques (6) (manual orautomatic), or simply by immerse plates in culture media then pull themout gently (dip-and-retract) (7), each reservoir will be filled withliquid thanks to capillary effect and liquid droplets will formautomatically due to surface tension force. Once the auxiliary platesare loaded with media, they are brought to a proper distance to a cellculture plate that also have convex liquid droplets formed at theopenings of each individual unit, the droplets are brought into contactand merge, they become small fluidic plugs (8) connecting reservoirswith their corresponding cell transfection units where cell reside. Forillustration purpose, only one feeding plate (1) is brought closely tothe cell culture plate (2) to form a fluidic connection. The bottomfeeding plate (2) is separated from the cell culture plate todemonstrate how the fluidic connection is formed by liquid droplets (5).Then the assembled device can be placed in cell culture incubator. Asthe thickness of the auxiliary medium feeding plate can be large,culture media reservoirs can hold enough media to feed cells forextended culture time. When cells are ready for electroporation, thecell cell culture plate is then separated from the cell culture platesfor downstream operations.

It should be noted that the medium feeding plate can also be used forparallel cell loading, which is demonstrated in FIG. 15. Using the same“dip-and-retract” approach as illustrated in FIG. 15 a, a feeding plate(1) is submerged in a solution (3) containing biological cells (4), thengently retracted. Due to surface treatment of the feeding platepreviously described, cell-containing solution fills in each opening ofthe feeding plates and forms convex liquid droplets (5) at the exits ofthe openings due to surface tension forces. A cell culture plate (2) isalso loaded with proper cell culture solution in each transfection unitsusing methods described previously. Convex liquid droplets (6) are alsoformed at the exits of each transfection unit due to surface tensionforces. When the feeding plate and the cell culture plate are broughtclose (FIG. 15 b), the liquid droplets (5 and 6) eventually merge toform a liquid connection (7), through which cells (4) in each opening ofthe feeding plate can travel to the corresponding transfection unit,thus completes cell loading process.

FIG. 16 illustrates another approach that uses disposable flexibleplates to increase cell culture volume. In this case, two flexibleplates (1) made of elastic inert materials (such as PDMS siliconerubber) are directly attached to a cell culture plate on both sidesthrough temporary bonding (FIG. 16 a). The inner surfaces (7) of eachopening of the flexible plate are treated to be hydrophilic, and therest surfaces (8) are hydrophobic. The two attached plates allow addingmore culture media (3) to feed the cells in each transfection unit of acell culture plate (2). In some special cases, cells might need to beculture for several weeks to reach a desirable biological state beforethey are transfected. For such expended long time of culturing, theentire assembly (cell cell culture plate and disposable plates) can beimmersed in a cell culture container, after cells attach to the porousmembrane of each transfection unit (normally takes 4-6 hours after cellsare introduced to the membrane surface). The attached plates also serveas protection layers to protect the hydrophobic-treated surfaces (6) ofthe cell culture plate from being exposed to and effected by cellculture media. When the cells are ready for electroporation andtransfection, the flexible auxiliary plates (1) are peeled off from thecell culture plate (2) (for illustration purposes, only the top flexibleplate is peeled off in FIG. 16 b). Excessive cell culture solution isalso automatically removed as it is retained inside the openings of theflexible plate due to surface tension force. The rest of the cellculture solution remains in each transfection units of the cell cultureplate, with convex droplets formed at the exits of the transfectionunits due to the protected hydrophobic surfaces (6), making them readyfor downstream downstream transfection procedures as describedpreviously.

Experimental Results Cell Culture

Various types of cells, including engineered cell lines (such as HepG2,MDCK, HEK293) and primary cells (such as HMEC, HUVEC, PrEC), are testedusing the described device. To culture cells in the cell culture device,cells are firstly trypsinized and resuspended in culture medium,followed by adding cells to a cell culture unit at seeding densitybetween 50,000 to 500,000 cells per cm² of porous membrane. Cell culturedevice is then placed inside an incubator at 37 C with 5% CO₂ for one ortwo days before they are electroporated and transfected. This practicetypically leads to formation of a >80% confluent cell monolayer on theporous membrane.

Electroporation Study

Electrical pulses with width between 100 msec to 5 sec are applied to apair of electrodes for cell electroporation. Depending on cell type andconfluence of cell monolayer, amplitude of electrical pulses ranges from1V to 3V for optimal electroporation of different types of cells. Anucleic acid staining fluorescent dye, YOYO-1 (Invitrogen), is used toassess the effective of electroporation. YOYO-1 molecules can not enternormal cells since they are membrane impermeant. When cells areelectroporated, YOYO-1 molecules can enter the cells through theirpermeablized membrane, and bind to cellular DNA and RNA molecules. Thus,the YOYO-1 containing electroporated cells can be readily identified asthey appear to be green under proper UV excitation. FIG. 17 shows MDCKcells that are electroporated in 1 uM YOYO-1 solution (in PBS) by a 2.5V/l second square pulse. To facilitate fluorescent observation, MDCKcells are detached from porous membrane and loaded on a glass slide. Inthe figure, bright cells indicates successful incorporation of membraneimpermeat YOYO-1 molecules due to electroporation. It can be seen >90%of the cells appear to be bright/white, indicating >90% of the cells areeffectively electroporated under that particular condition. Similarresults have also been achieved with other cells, including both celllines and primary cells, such as HEK293, HUVEC, PrEC, etc.

Transfection of DNA

FIG. 18 demonstrates the capability of the cell transfection device intransfection of cells with DNA plasmids. In the experiment, DNA plasmidssolution (10-50 ug/mL) is added either in the apical or basal side of acell monolayer, then cells are electroporated with square electricalpulses between 1.5V-3V. Expression of the DNA plasmids is observed fourto 24 hours after electroporation. It should be noted that using ourtransfection method, expression of DNA plasmids is evident as early asthree hours after transfection, which is significantly shorter thanother transfection methods (normally 24 hours). This rapid expression ofDNA molecules implies effective nuclear uptake of the DNA plasmids usingour method. FIG. 18 a shows successful GFP (Green Fluorescent Protein)expression in a confluent monolayer of PrEC cells (human prostateepithelial cells). More than 70% of the cells are successfullytransfected and express GFP, which appear bright in the image. FIG. 18 bshows GFP expression in a confluent monolayer of HMEC cells (humanmammary epithelial cells). More than 40% of the cells are successfullytransfected (bright cells in the picture). It should be noted that bothPrEC and HMEC are primary cells, which are typically very resistant toexisting transfection methods, such as lipofection. Being able totransfect primary cells in high-throughput fashion grants our technologyclear advantages over the existing ones.

Transfection of siRNA

To demonstrate our device's capability of transfecting cells with siRNA,we added 20 nM fluorescence labeled siRNA (Alexa Fluor 488-siRNA fromInvitrogen) to PrEC cells, then electroporated the cells using similarprotocols described above. FIG. 19 is an image showing effectiveintroduction of the siRNA molecules into more than 60% of cells (brightcells). To further demonstrate our capability, we first transfected PrECcells with GFP vectors. FIG. 20 a shows many bright cells indicatinghigh level GFP expression. Then we further transfected the same cellswith 10 nM anti-GFP siRNA (Invitrogen), which can knock down GFPexpression once they are delivered into cells. FIG. 20 b shows an imageof the same cells twelve hours after siRNA transfection. It is obviousthat cells appear much dimmer than before (FIG. 20 a), which indicatedsuccessful known-down of GFP in the cells by anti-GFP siRNA moleculesdelivered using our device.

Selective Electroporation and Transfection

To demonstrate the capability of performing selective electroporation,we used a UV curable transparent paste to block the edge region of theporous membrane in a cell transfection device. Cells are cultured on theother side of the porous membrane as previously described. Then thecells are electroporated in presence of a red nucleic acid stain,propidium iodine (PI), using similar conditions above. Then cells areinspected using fluorescent microscopy. FIG. 20 shows a fluorescentimage of MDCK cells after electroporation. The white dash line outlinesthe boundary of the cured transparent paste, which blocked the left sideof the porous membrane. MDCK cells formed a nice confluent monolayerspan across the entire porous membrane (image not shown). While as shownby the images, only the cells on the unblocked part of the porousmembrane (right side of the dash line) are successfully electroporated(indicated by PI uptake that make the cells appear to be bright in theimage). The cells on the blocked area (left side) were virtuallyunaffected since no PI uptake is detected (cells are dark and notvisible in the image).

Using the same method, we also performed selective cell transfectionsuccessfully in PrEC cells. GFP plasmids are used to indicate successfulcell transfection. FIG. 21 shows a fluorescent image of the cells.Similarly, a white dash line outlines the boundary between porousmembranes with blocked and open pores. Again, the experiment shows thatonly the cells on top of the porous membrane with unobstructed poresexpress GFP due to successful transfection (bright cells at the rightside of the dashline). The rest of the same cells under sameelectroporation conditions are virtually not affected. Both experimentsvalidate that selective electroporation and thus transfection can beachieved in our devices by blocking part of the porous membrane.

Methods of Use

The high-throughput cell transfection method disclosed in this inventioncan enable many cell-based assays for both fundamental research (such asfunctional genomics, cancer research) and therapeutic development (suchas target ID/validation, compound screen and in vitro testing).

Besides the general usage of the high-throughput transfection device andapparatus, we also disclose hereafter novel methods of using transienttransfected cells for evaluation and prediction of drug compounds'pharmaceutical and therapeutic properties. It should be noted that themethods to be disclosed can be realized by the aforementionedhigh-throughput cell transfection method and apparatus, however, theyare not necessarily limited to any particular transfection methods orapparatuses.

Method for Transcription Factor Activity Profiling

Transcription factors (TFs) are a family of regulatory proteins ofcritical importance as they control when genes are switched on or off.Knowing the functional activities of transcription factors not onlyoffer information on which genes will be transcripted (switched on), butalso can give insights on which signaling pathways are affected bystimuli (such as drugs, toxins, environmental stress, etc.) sinceactivation of transcription factors is normally the last step ofsignaling pathways. Furthermore, since it is the activation ofparticular set of transcription factors that turned on a particular setof genes that drive pathophysiological and physiological responses toexternal stimuli in human body, it is feasible to predict how human bodyresponses to a particular stimulus (drug, toxin, environmental stress,molecular defect, etc.) by analyzing changes in functional activities ofthe entire family of transcription factors, or a subset of pertinenttranscription factors.

The functional activity of a particular TF can be analyzed bytransfecting cells with a TF reporter vector that contains a cis-actingDNA response element upstream of the reporter gene. The reporter genecan encode any reporters such as luciferase, GFP, beta-lactamase, etc.The cis-reacting response element contains multiple repeats of aspecific TF binding element. When the vector is transfected in cells,activation of the specific transcription factor will result in bindingof the TF and the cis-acting element that recognize the TF, which causetranscription and expression of the reporter gene, producing adetectable signal as the result of activation of that particular TF.

To analyze the activities of a group of interested TFs in cells, it isnecessary to construct a library of TF reporter vectors, each reportervector contains cis-acting element that is recognized by a correspondingTF. Then one can use the transfection device previously described, oruse any other applicable methods, to transfect a number of the same cellsamples with the TF reporter vectors, one vector for each sample. Eachtransfected cell sample is used to elucidate the functional activity ofone TF, collectively, one can profile the activities of a large numberof TFs in a particular type of cells (primary cells or engineered celllines).

When the cells are subject to a stimulus, comparing the functionalactivity profiles of a particular set of TFs in cells before and afterstimulation can reveal which TFs' activities are affected by thestimulus (drug, toxin, stress, radiation, molecular change, etc.) Thiscan be particularly useful to evaluate the effects of the stimulus andto understand the underlying mechanisms that cause the actions(Mechanism-Of-Action).

It should be noted that in order to compare TFs' functional activitiesin the same cells before and after stimulation, the reporter used mustpermit analysis of the reporter activity in live cells. Such reportercan be GFP, renilla luciferase, beta-lactamase, etc. If the reporterchosen requires lysing cells (e.g.; firefly luciferase), two sets ofexperiments need to be carried out, with one set of transfected cellsnot being treated with stimulus to serve as negativecontrols/references, the other set of transfected cells to be treatedwith stimulus in order to obtain the corresponding TF activity profile.The changes in TF activities can be extrapolated by comparing the twoprofiles obtained from different sets of cells.

To recapitulate, the method for profiling the changes in functionalactivities of a set of transcription factors caused by a stimuluscomprise typically the following steps,

-   -   a. Transfecting at least one type of model cells (primary cells        or engineered cell lines) with a library (>2) of TF reporter        vectors, with one population of cells transfected with one TF        reporter vector that can recognize functional activity of a        specific TF inside the cells.    -   b. Measure reporter activities through fluorescence,        luminescence or any appropriate techniques, in each population        of cells, to evaluate the functional activity of the specific TF        corresponding to the IF reporter vector being transfected in        this particular population of cells.    -   c. Construct a profile of the functional activities of all TFs        of interests    -   d. If the reporter system used allows live cell analysis,        subject the previous cells to a specific stimulus, repeat step        a-c, to obtain a second profile of TF activities in the same        cells after stimulation. If the reporter used doesn't allow live        cell analysis, start with a new batch of same model cells,        subject the cells to the stimulus, then go through step i-iii to        obtain a second profile of TF activities in the model cells that        underwent stimulation.    -   e. Compare the first and second profiles to identify changes in        functional activities of the TFs the type(s) of model cells        caused by the stimulus

Target Evaluation and Biomarker Discovery Using TF Activity Profiling

Modern drugs often modulate target proteins that play important roles inpathological pathways. Direct or indirect modulation of the targetsoften result in changes in activities of many expected and unexpectedtransfection factors because of cross-talking among signaling pathways.The aforementioned TF activity profiling method can be used to evaluatetherapeutic and side effects caused by modulating a particular drugtarget, as well as to discover biomarkers that serve as a surrogatemeasure on the effectiveness of target modulation.

A method using transcription factor activity profiling for drug targetevaluation and or biomarker discovery is disclosed. The method comprisestypically the following steps:

-   -   a. Transfecting at least one type of model cells (primary cells        or engineered cell lines) with a library (>2) of TF reporter        vectors, with one population of cells transfected with one TF        reporter vector that can recognize functional activity of a        specific TF inside the cells.    -   b. Measure reporter activities through fluorescence,        luminescence or any appropriate techniques, in each population        of cells, to evaluate the functional activity of the specific TF        corresponding to the TF reporter vector being transfected in        this particular population of cells.    -   c. Construct a profile of the functional activities of all        interested TFs    -   d. If the reporter system used allow live cell analysis, treat        the previous cells using appropriate methods to modulate a drug        target of interests, repeat step i-iii, to obtain a second        profile of TF activities in the same cells that are after drug        target modulation. If the reporter used doesn't allow live cell        analysis, start with a new batch of cells, apply appropriate        methods to modulate the drug target of interests in these cells,        then go through step i-iii to obtain a second profile of TF        activities in cells that underwent target modulation.    -   e. Compare the first and second profiles to identify changes in        functional activities of the TFs the type(s) of model cells due        to modulating the said target

Once the changes in TFs' functional activities are elucidated, one canfurther identify which pathways the drug target by identifying theaffected TFs and the pathways they are linked to. One can also furtherpredict the consequences of modulating this drug target by analyzing howthe changes in TF activities would influence transcription andexpression of relevant genes, and furthermore what therapeutic and toxiceffects the changes in expression of the genes would lead to. Both theaffected TFs and the downstream genes and proteins due to modulation ofthe drug target can be used as biomarkers, which can be used inpreclinical and/or clinical studies as indications to effectiveness oftherapeutic intervention and/or clinical safety.

Drug Evaluation Using TF Activity Profiling

Modern drugs are often designed to regulate one particular protein (drugtarget) with important roles in a pathological pathway. Most times, thedrugs have in-target effects, which are the consequences of a successfulmodulation of the drug target (structurally or functionally), andoff-target effects due to drugs' unintended influence on other proteins.Both the in-target and off-target effects often result in activation ofa number of TFs mediated by signaling transduction. By analyzing thechanges in TF activities in appropriate model cells before and afterdrug compounds treatment, it is possible to elucidate the therapeuticand side-effects of the drug compounds.

A method that uses transcription factor activity profiling for drugcompound evaluation is disclosed. The method comprises typically thefollowing steps:

-   -   a. Transfecting at least one type of model cells (primary cells        or engineered cell lines) with a library (>2) of TF reporter        vectors, with one population of cells transfected with one TF        reporter vector that can recognize functional activity of a        specific TF inside the cells.    -   b. Measure reporter activities through fluorescence,        luminescence or any appropriate techniques, in each population        of cells, to evaluate the functional activity of the specific TF        corresponding to the TF reporter vector being transfected in        this particular population of cells.    -   c. Construct a profile of the functional activities of all        interested TFs    -   d. If the reporter system used allow live cell analysis, treat        the previous cells with the drug compound(s) of interests using        appropriate methods, repeat step i-iii, to obtain a second        profile of TF activities in the same cells that are after        compound treatment. If the reporter used doesn't allow live cell        analysis, start with a new batch of cells, treat these cells        with the drug compounds) of interests using appropriate methods,        then go through step i-iii to obtain a second profile of TF        activities in cells underwent compound treatment.    -   e. Compare the first and second profiles to identify changes in        functional activities of the TFs in the type(s) of model cells        due to treatment with the said drug compound(s)

Once changes in TF activities due to drug treatment are revealed, onecan further identify which pathways were affected by the compound(s) byidentifying the affected TFs and the pathways they are linked to. Onecan also further predict the consequences of compound treatment byanalyzing how the changes in TF activities would influencetranscription, thus expression of relevant genes, and furthermore whattherapeutic and toxic effects the changes in transcription of thedownstream genes would lead to. Both the TFs and the downstream genesand proteins affected by the drug treatment can be used as biomarkers toindicate to effectiveness of therapeutic intervention and/or clinicalsafety in preclinical and/or clinical studies.

Method to Predict Effects of a Stimulus in Human

By combining the transcription factor activity profiling method andbioinformatic techniques, we propose a novel method to predicttherapeutic and toxic/safety effects of a stimulus in human. Thestimulus can be exposure to chemicals, therapeutic agents and toxins,radiation, molecular intervention such as modulation of a drug target,and any other perturbations that can cause molecular and cellularresponses by biological entities, including human and animals. Themethod comprises typically the following steps,

Obtain changes in functional activities of at least 2 TFs in at leastone type of model cells caused by a member of a set of reference stimuliwhose effects in human are well characterized (such stimuli can beapproved drugs, known toxins, etc.), using the method describedpreviously.

-   -   a. Correlate the changes in the functional activities of the        said TFs in the said model cells, with the known therapeutic and        toxic/safety effects of the said reference stimuli in human    -   b. Obtain changes in the functional activities of the said TFs        in the same model cells caused by a test stimulus whose effects        in human are not well characterized (such stimulus can be a new        drug in development, a new toxin, etc.), using the method(s)        described previously in section 2.b.    -   c. Compare the changes in TFs' functional activities in the said        model cells caused by the test stimulus and the changes in TF        functional activities caused by the reference stimuli, identify        possible similarities among the changes, correlate the        similarities with the therapeutic and toxic/safety effects of        the reference stimuli in human to predict the therapeutic and        toxic/safety effects of the test stimulus in human.

Method for Determining Transporter Proteins Influencing DrugTransportation

Transporter proteins are a family of membrane proteins that areresponsible for transporting a variety of substances (small moleculedrugs, ions, peptides, etc.) in and out of cells. They play key roles indrug absorption, distribution and excretion. Being able to profile whichtransporter proteins affect transportation of a drug compound in and outof cells can be very useful in characterizing the compound's ADME orpharmacokinetic properties and in providing mechanistic insights on howto improve the compound's ADME/PK properties.

A commonly used in vitro method to study transportation of a compound isto study how the test compound is transported through a cell monolayergrown on a porous membrane. This transcellular transport analysis methodcan be readily realized using the previously disclosed transfectiondevices that allow formation of confluent cell monolayers on a porousmembrane inside each transfection unit. By combining cell transfectionand transcellular transport analysis, we come up with a novel method toevaluate influence of individual transporters on a test compound'sADME/PK properties.

The method comprises typically the following steps,

-   -   a. Grow an appropriate model cells into confluent monolayers on        porous membranes in an array of applicable devices. The model        cells must be able to grow into confluent monolayer with tight        junctions formed among adjacent cells, and the model cells have        low native expression level of transporter proteins. MDCK cell        line is a good candidate as it meets both requirements.    -   b. Transfect the cells with a library of DNA plasmids using        applicable methods. The cells in each device are transfected        with one type of DNA plasmids that encode a specific transporter        protein. As a result, an array of testing cell monolayers is        created with each cell monolayer expressing a particular        transporter protein.    -   c. Use applicable analytical methods to measure a test        compound's permeability to every cell monolayer expressing the        known transporter protein, compare with the reference        permeability of the said compound to the wild-type model cells        to identify all the individual transporter proteins that affect        the permeability of the said compound to the model cell        monolayers and the relative significance of their influence.

Once the individual transporter proteins that affect the compound'stransportation are identified, one can analyze which individualtransporter-compound interaction improve and which deteriorates thecompound's ADME/PK properties. This information can be very valuable toguide compound optimization to modify compound structure so that itbecomes the substrate of particular transporter proteins to render thecompound desirable ADME/PK properties or therapeutic effects. Forexample, many influx transporters such as PEPT1, ASBT, OATP-B, etc. helpimprove drug absorption, while efflux transporters such as P-gp, MRP2and BCRP play the opposite role. Therefore, if a compound is identifiedto the substrate of particular efflux transporters, one can modify thecompound structure to avoid binding to the efflux transporters,furthermore, to transform the compound as the substrate of some influxtransporters, thus improving the absorption properties of the drugcompound.

Another example is to optimize compound structure for targeted drugdelivery. The strategy is to focus on differential expression oftransporters between the target cells/organ and other cells/organs. Theinformation obtained from compound-transporter interaction profiling canhelp guide structural optimization for compound so that it is absorbedonly by the target cells/organ.

Another valuable application of the method is in drug toxicityprediction. There are many transporters only expressed in particularorgans, for example, NTCP is a transporter exclusively expressed inliver, a drug compound that is the substrate of NTCP transporter is morelikely to have liver toxicity due to the high chance of being deliveredto liver. Similarly, a drug that is the substrate of blood-brain barrier(BBB)-specific transporters is more likely to penetrate BBB to reachbrain, thus has higher possibility of causing neurotoxicity.

Method to Predict Compound's ADME/PK Properties

ROM By combining the compound-transporter interaction profiling methodand bioinformatic techniques, we propose a novel method to predict acompound's ADME/PK properties. The'method comprises typically thefollowing steps,

-   -   a. Obtain the profiles on the interactions of each member of a        reference compound library with a library (>2) of transporter        proteins in at least one type of model cells, using the method        described previously in section 2.e. The reference compounds are        approved or failed drugs whose ADME/PK properties in human are        well characterized.    -   b. Correlate the compound-transporter interaction profiles in        the said model cells, with the ADME/PK properties of the said        reference compounds    -   c. Obtain the profile on the interactions of a test drug        compound with the library of transporter proteins in the same        model cells, using the method described previously in section        2.e.    -   d. Compare the profiles on transporter interaction in the said        model cells among the test compound and the reference compounds,        identify possible similarities, correlate the similarities with        the ADME/PK properties of the reference compounds in human to        predict the ADME/PK properties of the test compound in human.

1. A method of identifying a transporter protein that affects transportof a compound in and/or out of a cell, comprising: a. providing multiplepopulations of cells, wherein upon transient transfection with adistinct foreign substance, each population of cells have alteredexpression of a distinct transporter protein as compared to other cellpopulations; b. assaying the multiple populations of cells as early asthree hours after said transient transfection for the ability of saiddistinct transporter expressed in said each population of cells totransport the compound in and/or out of a cell.
 2. The method of claim1, wherein the step of providing comprises (i) culturing each populationof cells in a distinct chamber supported by a porous membrane, and (ii)transiently transfecting said each population of cells with a vectorencoding the distinct transporter protein by way of electroporation. 3.The method of claim 1, wherein the step of providing comprises (i)culturing each population of cells in a distinct chamber supported by aporous membrane, and (ii) transiently transfecting said each populationof cells with a siRNA targeting the distinct transporter protein by wayof electroporation.
 4. The method of claim 1, wherein the transporterprotein is selected from the group consisting of PEPT1, ASBT, OATP-B,P-gp, MRP2, and BCRP.
 5. The method of claim 1, wherein the transporterprotein is an efflux transporter.
 6. The method of claim 1, wherein thetransporter protein is an influx transporter.
 7. The method of claim 1,wherein the ability of said distinct transporter expressed in said eachpopulation of cells to transport the compound in and/or out of a cell isevidenced by a fluorescent or luminescent readout.
 8. The method ofclaim 1, wherein the multiple populations of cells are of the same type.9. The method of claim 1, wherein the multiple populations of cells areof different types.
 10. The method of claim 1, wherein the cells areadherent.
 11. The method of claim 1, wherein the cells are monolayercells.
 12. The method of claim 1, wherein the cells are polarized. 13.The method of claim 1, wherein the cells are selected from the groupconsisting of prostate cells, mammary cells, kidney cells,blood-brain-barrier cells, and liver cells.
 14. The method of claim 1,wherein the cells are cancer cells.
 15. The method of claim 1, where thecells are selected from the group consisting of MDCK (Madin-Darby CanineKidney) cells, HEK293, and HepG2.
 16. The method of claim 1, wherein thecells are primary cells directly derived from tissues.
 17. The method ofclaim 1, wherein the cells are epithelial cells.
 18. The method of claim1, wherein the assaying of multiple populations of cells is carried outwithin 6, 12, 24, 48 or 72 hours.
 19. An in vitro method of assaying fortransport of a test compound in and/or out of a cell, comprising: a.transiently transfecting a monolayer of cells adhered to a porousmembrane with a foreign substance suspected to affect transport of saidtest compound, wherein said transfection results in at least about 40%of the cells of said monolayer being transfected with said foreignsubstance; b. incubating said transfected monolayer of cells with saidtest compound; and c. measuring an effect of said foreign substance insaid cells on the transport of said test compound.
 20. The method claim19, wherein the monolayer of cells comprises polarized cells.
 21. Themethod of claim 19, wherein the transporter protein is an effluxtransporter.
 22. The method of claim 19, wherein the transporter proteinis an infflux transporter.
 23. The method of claim 19, wherein the cellsare selected from the group consisting of prostate cells, mammary cells,kidney cells, blood-brain-barrier cells, and liver cells.
 24. The methodof claim 19, wherein the cells are cancer cells.
 25. The method of claim19, where the cells are selected from the group consisting of MDCK(Madin-Darby Canine Kidney) cells, HEK293, and HepG2.
 26. The method ofclaim 19, wherein the cells are primary cells directly derived fromtissues.
 27. The method of claim 19, wherein the cells are epithelialcells.
 28. The method claim 19, wherein at least about 70% of themonolayer cells are transfected with the foreign substance.
 29. Themethod claim 19, wherein at least about 90% of the monolayer cells aretransfected with the foreign substance.
 30. The method of claim 19,wherein the foreign substance comprises a vector encoding one or moretransporter proteins.
 31. The method of claim 19, wherein the foreignsubstance is selected from the group consisting of DNA, siRNA, protein,antibody, peptide, chemical compound, and a nanoparticle.
 32. The methodof claim 19, wherein the step of measuring is conducted within 6, 12,24, 48 or 72 hours after said transient transfection.
 33. The method ofclaim 19, wherein the test compound is a drug.
 34. A method ofpredicting a pharmacological property of a test compound, comprising: a.transiently transfecting multiple populations of cells, each populationof which, with a distinct foreign substance to affect expression of adistinct transporter protein; b. identifying from said transientlytransfected populations of cells the transporter proteins that affecttransport of test compound to generate a test compound/transporterinteraction profile; c. comparing said test compound/transporterinteraction profile with that of a reference compound whosecompound/transporter interaction profile and correspondingpharmacological property are characterized, thereby predicting thepharmacological property based on said comparison.
 35. The method ofclaim 34, further comprising modifying the structure of the testcompound such that the test compound yields an altered testcompound/transporter interaction profile as compared to that of the testcompound without the modification.
 36. The method of claim 34, whereinthe pharmacological property comprises ADME and pharmacokineticproperties.
 37. The method of claim 34, wherein the cells are adherent.38. The method of claim 34, wherein the cells are adherent monolayercells.
 39. The method of claim 34, wherein the cells are polarized. 40.The method of claim 34, wherein the transporter protein is selected fromthe group consisting of PEPT1, ASBT, OATP-B, P-gp, MRP2, and BCRP. 41.The method of claim 34, wherein the reference compound is an approved orfailed drug.